Bone marrow is the home for hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs). MSCs have demonstrated their promise in the repair or regeneration of bone [1, 2], cartilage [3, 4], tendon [5], and meniscus [6]. Bone marrow provides a tissue-specific microenvironment, also known as a niche, for the MSCs. This microenvironment is created by a variety of stromal cells [7, 8]. Our recent study indicated that MSCs cultured on marrow stromal-cell derived extracellular matrix (ECM) dramatically promoted MSC proliferation, preserved the MSC properties, and enhanced their capacity for skeletogenesis [9]. There is an accumulating body of evidence suggesting that the tissue-specific environment and its temperature, oxygen tension and perfusion flow all act together to promote and regulate the self-renewal, commitment, differentiation, and proliferation of the MSCs [13]. The mechanism of how the bone marrow microenvironment regulates the behavior of MSCs, however, is unclear. In vitro three-dimensional (3D) analogs of human bone marrow have been developed to study cell behavior in bone marrow [14-17]. However, these 3D models, prepared from a single component of either a synthetic material or a purified protein, do not faithfully represent the tissue-specific bone marrow environment. These models also lack control of temperature, oxygen tension, and perfusion rate, which have shown their importance in the regulation of MSCs in bone marrow. The objective of this proposal is to reconstitute a native tissue-specific bone marrow environment with the control of temperature, oxygen tension, and perfusion rate for a more faithful simulation of the bone marrow environment where MSCs reside in vivo. To achieve this objective, we propose to develop a tissue-specific 3D environment using a cross-linked collagen scaffold that mimics the trabecular-bone's porous architecture and its surfaces will be "coated" with a native bone marrow stromal cell-derived ECM. A perfusion flow bioreactor will be developed to provide a uniform nutrition supply to the tissue-specific scaffolds and a precise control of perfusion flow, temperature, O2 and CO2 tensions. The in vitro system will be tested by MSC viability and distribution assays. The MSC proliferation will be evaluated under different culture conditions of temperature, oxygen tension, and flow rate. The establishment of this in vitro bone marrow model will provide a tool necessary for the study of cell-cell, cell-matrix interactions, MSC migration, the growth factors and cytokines that regulate MSC self-renewal and mutilineal differentiations, and other MSC activities in the bone marrow environment. The knowledge learned from these studies is important for the development of an in vitro MSC niche. This system also has the potential to be developed into an in vitro expansion device that provides a continuous source of blood cells for transplantation and MSCs for tissue regeneration. PUBLIC HEALTH RELEVANCE: This application aims to reconstitute an in vitro bone marrow system for the study of mesenchymal stem cells and their niche in vivo. The ex vivo bone marrow system will include a bone-marrow tissue-specific three-dimensional scaffold to mimic the trabecular bone and bone marrow tissue, and a bioreactor system to simulate the perfusion flow, temperature, and oxygen tension in the in vivo bone marrow environment. The establishment of this system will provide a tool necessary for the study of cell-cell, cell-matrix interaction, MSC migration, the growth factors and cytokines that regulate MSC self-renewal and mutilineal differentiations, and other MSC activities in the bone marrow environment. The knowledge learned from these studies is important for the development of an in vitro MSC niche. This system also has the potential to be developed into an in vitro expansion device that provides a continuous source of blood cells for transplantation and MSCs for tissue regeneration.